Synthesis, Spectral Characterization, In-vitro Antibacterial and Antifungal Activities of Novel (2e)-Ethyl-2-(2-(2, 4-Dinitrophenyl) Hydrazono)-4-(Naphthalen-2-yl)-6-Arylcyclohex-3-Enecarboxylates.

In a search for new leads towards potent antimicrobial agents, an array of novel (2E)-ethyl-2-(2-(2,4-dinitrophenyl)hydrazono)-4-(naphthalen-2-yl)-6-arylcyclohex-3-ene carboxylates 17-24 were synthesized and characterized through their melting point, elemental analysis, MS, FT-IR, one-dimensional NMR (1H, D2O exchanged 1H and 13C), two dimensional HOMOCOR and HSQC spectroscopic data. In-vitro microbiological evaluations were carried out for all the newly synthesized compounds 17-24 against clinically isolated bacterial strains namely Salmonella typhii, Klebsiellapneumoniae, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, β-Hemolytic streptococcus and Micrococcus luteusand also fungal strains namely Aspergillusflavus, Aspergillusniger, Mucor, Rhizopus and Microsporumgypseumand finally, the results of their structure activity relationship were discussed. The obtained results can be used as the key step for the building of novel chemical compounds with interesting antimicrobial profiles comparable to that of the standard drugs.


Introduction
Over the past few decades, health-related quality of human life benefits are under threat as many commonly used antibiotics have become less and less effective against certain illnesses, not only because many of them produce toxic reactions but also due to emergence of drug resistant microbes. Hydrazones constitute an important class of compounds for new drug development. Therefore, many researchers have synthesized these compounds as target structures and evaluated their biological activities. These observations have been conducted for the development of new hydrazones that possess varied biological activities (1). Some critical reviews have been published which give an outlook on the developments on antimycobacterial hydrazones (2)(3)(4)(5). Hydrazones have been demonstrated to possess varied biological activities (Figure1). For example, isonicotinoyl hydrazones (A) are antitubercular and nifuroxazide (B) is an intestinal antiseptic. Acetylhydrazones (C) provided a good protection against convulsions (6).
The 2-Acetyl naphthalene is an interesting starting material which is widely used in perfume formulations (15), mainly in Neroli orange blossom, sweet pea, magnolia, honeysuckle, wisteria, narcisse, jasmine, various exotic florals, etc. In flavor composition, also, the ketone finds a place in imitation of strawberry, grape, various citrus and berry compositions in neroli and other natural flavors, in fruit complexes and in certain types of vanilla flavor. Nowadays, there has been a great deal of interest in exploiting more than one proximal functional groups for designing novel structures capable of performing a variety of functions. Synthesis of molecules, which are novel yet resembling known biologically active molecules by virtue of the presence of some critical structural features, is an essential component of the search for new leads in drug designing programme. Taking these considerations into account and as a part of our research program aimed at the synthesis of bioactive novel structurally diverse heterocycles (16-20), herein is reported the molecular conjugation of the naphthyl substituted chalcones moiety with two or more active counterparts which has been designed and synthesized with the hope of producing novel ethyl 4-(naphthalen-2-yl)-2-oxo-6-arylcyclohex-3-enecarboxylates (10-17), an intermediate with three versatile functional groups i.e., ketone, olefin and ester for the synthesis of (2E)-ethyl-2-(2-(2, 4-dinitrophenyl)hydrazono)-4-(naphthalen-2yl)-6-arylcyclohex-3-ene carboxylates 17-24, a novel hydrazone derivative and to study their in-vitro microbiological evaluation against clinically isolated bacterial and fungal strains.

Chemistry
The progress of the reaction is monitored by TLC analysis. All the reported melting points are taken in open capillaries and are uncorrected. IR spectra are recorded in KBr (pellet forms) on a Nicolet-Avatar-330 FT-IR spectrophotometer and note worthy absorption values (cm -1 ) alone are listed. 1 H and 13 C NMR spectra are recorded at 400 MHz and 100 MHz respectively on Bruker Avance II 400 NMR spectrometer using DMSO-d 6 as solvent. Two dimensional HOMOCOR and HSQC spectra are recorded at Bruker DRX 500 NMR spectrometer. The ESI +ve MS spectra are recorded on a Bruker Daltonics LC-MS spectrometer. Satisfactory microanalyses are obtained on Carlo Erba 1106 CHN analyzer.

In-vitro antibacterial and antifungal activity
The minimum inhibitory concentration (MIC) of μg/mL values is carried out by two-fold serial dilution method (22). The respective test compounds 17-24 are dissolved in dimethyl sulfoxide (DMSO) to obtain 1 mg mL -1 stock solution. Seeded broth (broth containing microbial spores) is prepared in NB from 24 h old bacterial cultures on nutrient agar (Hi-media, Mumbai) at 37 ± 1 °C while fungal spores from 1 to 7 days old Sabouraud agar (Hi-media, Mumbai) slant cultures were suspended in SDB. The colony forming units (cfu) of the seeded broth are determined by plating technique and adjusted in the range of 10 4 -10 5 cfu/mL. The final inoculums size was 10 5 cfu/mL for antibacterial assay and 1.1-1.5 X 10 2 cfu/mL for antifungal assay. Testing is performed at pH of 7.4 ± 0.2 for bacteria (NB) and at a pH of 5.6 for fungi (SDB). Exactly 0.4 mL of the solution of test compound was added to 1.6 mL of seeded broth to form the first dilution. One mL of this was diluted with a further 1 mL of seeded broth to give the second dilution and so on till six of such dilutions are obtained. A set of assay tubes containing only seeded broth was kept as control. The tubes were incubated in BOD incubators at 37 ± 1°C for bacteria and 28 ± 1°C for fungi. The minimum inhibitory concentrations (MICs) are recorded through visual observations after 24 h (for bacteria) and 72-96 h (for fungi) of incubation. Ciprofloxacin is used as standard for bacterial studies and Fluconazole is used as standards for fungal studies.

Scheme-2. Mechanistic pathway for the formation of ethyl 4-(naphthalen-2-yl)-2-oxo-6-phenylcyclohex-3-enecarboxylates and (2E)-ethyl 2-(2-(2,4-dinitrophenyl)hydrazono)-4-(naphthalen-2-yl)-6-arylcyclohex-3-enecarboxylates
FT-IR spectrum of (2E)-ethyl-2-(2-(2,4dinitrophenylhydrazono)-4-(naphthalen-2-yl)-6-phenylcyclohex-3-enecarboxylate 17 shows characteristic absorption frequency at 3323 cm -1 which suggests the presence of -NH functional group. The absorption frequency at 1656 cm -1 is assigned to C=N stretching vibration. The band at 1512 cm -1 is due to the presence of C=C stretching frequency. Nitro functional groups show characteristic stretching frequency around 1330 cm -1 . Besides these, aromatic CH stretching frequencies are observed at 3099 and 3060 cm -1 and the aliphatic CH stretching frequencies are observed at 2989, 2924 and 2847 cm -1 . The observed -NH, C=N, C=C and NO 2 stretching vibrational bands are supporting evidence for the formation of synthesized compound 17. In the 1 H NMR spectrum of 17, a triplet was observed at 0.92 ppm (J = 7.0 Hz) corresponding to three protons and this signal is due to ester methyl protons at C-1. A quartet observed at 3.91 ppm (J = 7.0 Hz) corresponding to two protons and this signal is due to ester methylene protons at C-1. Two multiplets are obtained in the range 3.20-3.21 and 3.66-3.72 and they are due to H-5 and H-6 protons. The doublet at 4.16 ppm (J = 13.4 Hz) has been assigned to H-1 proton. The singlet observed in downfield region at 6.73 ppm is due to H-3 proton. The aromatic protons appeared as a multiplet in the range of 7.25-8.80 ppm. The aromatic protons due to dinitrophenyl part are observed in the range of 8.12-8.80 ppm. H-3 proton of 2,4-dinitrophenyl part gives signal as a doublet at 8.80 ppm (J = 2.5 Hz). H-5 proton of 2,4-dinitrophenyl part gives signal as a multiplet around 8.32-8.23 ppm. H-6 proton of 2,4-dinitrophenyl part gives signal as a doublet at 8.12 ppm (J = 7.3Hz). The labile NH proton (exchangeable with D 2 O) appears as a broad singlet at 9.97 ppm. In the HOMOCOSY spectrum of 17, the signal at 0.92 ppm shows cross peak with the signal at 3.91 ppm and vice versa. From the above cross peaks, it is confirmed that the triplet observed at 0.92 ppm corresponds to ester methyl protons at C-1, whereas the quartet at 3.91 ppm corresponds to ester methylene protons at C-1. The signal at 4.16 ppm gives cross peak with multiplet at 3.66-3.72. Similarly, the multiplet at 3.66-3.72 gives cross peaks with the signal at 4.16 ppm as well as the signal at 3.20-3.21 ppm. Furthermore, the signal at 6.73 ppm gives correlations with the signal at 3.20-3.21 ppm and vice versa. From the above correlations, it reveals that the singlet at 6.73 ppm corresponds to H-3 proton and the multiplet at 3.66-3.72 ppm corresponds to H-6 proton respectively. The doublet at 4.16 ppm is conveniently assigned to H-1 proton and the multiplet at 3.20-3.21 ppm is unambiguously assigned to H-5 proton.
In the 13 C NMR spectrum of 17, five resonances in the aliphatic region at 13.8, 35.1, 43.8, 58.7 and 59.9 ppm are observed. They are all due to CH 2 CH 3 at C-1, C-5, C-6, C-1 and CH 2 CH 3 at C-1 respectively. C-3 carbon resonates at 115.5 ppm. The remaining 13 C resonances in quaternary carbon signals at 169.2 and 158.7 ppm are due to C=O at C-1 and C-2 carbon respectively.
The aromatic carbons are observed in the range of 123.1-128.8 ppm. The carbon signals due to aromatic carbons of 2,4-dinitrophenyl part are merged with the aromatic carbons of naphthyl and phenyl rings at the position of 4 and 6 in cyclohexene ring. C-4 carbon may be merged with aromatic carbon signals. The 13 C resonances at 129.5, 132.7, 133.6, 134.3, 141.5 and 149.5 ppm are due to ipso carbons. In the HSQC spectrum of 17, one bond correlation (13.8/0.92 ppm) is observed between CH 2 CH 3 at C-1 and CH 2 CH 3 at C-1. The 13 C resonance at 59.9 has correlations with ester methylene protons, CH 2 CH 3 at C-1. Another one-bond correlation (35.1/3.20-3.21 ppm) is observed between C-5 and H-5. The 13 C resonance at 43.8 ppm has a one-bond correlation with a multiplet around 3.66-3.72 ppm. Hence, the signal at 43.8 ppm corresponds to C-6 carbon whereas the multiplet at 3.66-3.72 ppm is assigned to H-6 proton. The other aliphatic carbon which resonances at 58.7 ppm, shows a one-bond correlation with a doublet at 4.16 ppm. From this correlation, it is revealed that the doublet at 4.16 ppm corresponds to H-1 proton of the cyclohexenone moiety and the 13 C signal at 58.7 is assigned to C-1 carbon. The 13 C resonance at 115.5 ppm has correlations with singlet at 6.73 ppm. So the signal at 6.73 ppm is conveniently assigned to H-3 proton and the carbon signal at 115.5 ppm is assigned to C-3. In the HSQC, the 13 C resonances at 169.2 and 158.7 ppm has no correlations with protons and hence it is due to quaternary carbons, ester C=O at C-1 and C=N respectively. The C-4 carbon may be merged with the aromatic carbons. Among the quaternary carbons, the 13 C resonances at 129.5, 132.7, 133.6, 134.3, 141.5 and 149.5 are due to ipso carbons.

Antibacterial activity
Novel (2E)-ethyl-2-(2-(2,4-dinitrophenyl) h y d r a z o n o ) -4 -( n a p h t h a l e n -2 -y l ) -6arylcyclohex-3-enecarboxylates 17-24 are tested for their antibacterial activity in-vitro against S. typhi, K.pneumoniae, E.coli, P.aeruginosa, S.aureus, β-H.streptococcus and M.luteus. Ciprofloxacin is used as standard bactericidal drug. Minimum inhibitory concentration (MIC) in μg/mL values is reproduced in Table 2. A close survey of the MIC values indicates that all the compounds 17-24 exhibited a varied range (6.25-200 μg/mL) of antibacterial activity against all the tested bacterial strains except compounds 18 and 20 against E.coli and K.pneumoniae respectively which do not have activity even at a maximum concentration of 200 μg/mL. Compounds 18 and 20, which have electron donating methyl/methoxy substituent at the para position of phenyl rings attached to C-6 of cyclohexenone moiety, showed moderate activity against Gram-negative bacteria and showed good activity against Gram-positive bacteria at a MIC value in the range of 6.25-12.5 μg/mL against S. aureus, β.H.streptococcus and M. luteus. Compound 17 which has no substitution at the phenyl ring attached to C-6 of cyclohexenone moiety, exhibited moderate activity against all the tested bacterial strains. Compound 19 shows excellent antibacterial activity against S. typhi, K.pneumoniae, P.aeruginosa and β.H.streptococcus at a MIC value of 6.25 μg/mL which is a four-fold increase in activity with that of Ciprofloxacin. Similarly, compound 21 which has chloro substitution at the para position of phenyl rings attached to the C-6 of cyclohexenone moiety, exerted excellent activity against E.coli and M. luteus at a MIC value of 6.25 μg/mL and exerted a MIC value of 12.5 μg/mL against S. typhi, P.aeruginosa and S. aureus respectively. In addition, compound 24, a meta chloro substituted compound exhibited good antibacterial activity against K. pneumoniae and S. aureus at a MIC value of 12.5 μg/mL whereas it showed activity at a MIC of 6.25 μg/mL against β.H.streptococcus. Bulky bromo substitution at the para position of phenyl rings attached to C-6 of cyclohexenone moiety in compound 22 is active against P. aeruginosa. Moreover, compound 23 which has nitro substitution at the para position of phenyl rings attached to C-6 of cyclohexenone moiety exerted excellent activity against P. aeruginosa and S. aureus at a MIC value of 6.25 μg/mL and it showed good activity against K. pneumonia at a MIC value of 12.5 μg/mL. All the electron withdrawing substituent namely fluoro, chloro, bromo or nitro compounds such as 19, 21-24 exerted strong antibacterial activity against all the tested strains when compared to standard drug Ciprofloxacin whereas electron donating substituent namely methyl or methoxy compounds such as 18 and 20 show good activity against Gram-positive bacteria at a MIC value in the range of 6.25-12.5 μg/mL.

Antifungal activity
The in-vitro antifungal activity of (2E)ethyl-2-(2-(2,4-dinitrophenyl)hydrazono)-4 -( n a p h t h a l e n -2 -y l ) -6 -a r y l c y c l o h e x -3enecarboxylates 17-24 was studied against the fungal strains viz., A. flavus, A. Niger, Mucor, Rhizopus and Microsporum gypseum. Fluconazole was used as a standard drug. Minimum inhibitory concentration (MIC) in μg/mL values is reproduced in Table 3. A close survey of the MIC values indicates that all the compounds 17-24 exhibited a varied range (6.25-200 μg/mL) of antifungal activity against all the tested fungal strains except compounds 17, 21 and 23 which do not have antifungal activity against M. gypseum, Rhizopus and A. flavus respectively even at a high concentration of 200 μg/mL. Compound 17, having no substitution at the phenyl rings attached to C-6 carbon of cyclohexenone moiety exerted moderate activity against all the tested fungal strains, whereas methyl substituted compound 18 exerted good antifungal activities against Mucor, Rhizopus at a MIC value of 6.25 and 12.5 μg/mL respectively. Similar results are noticed for electron donating methoxy substituent compound 20 and exerted antifungal activity against Rhizopus at a MIC of 6.25 μg/mL, whereas it showed potent activity against A. Niger and Mucor at a MIC of 12.5 μg/mL. Compound 19 showed excellent antifungal activity against all the tested fungal strains and it showed MIC value of 6.25-25 μg/ mL. Similarly, chloro substituted compound 21 and nitro substituted compound 23 exhibited good antifungal activities against all the tested strains except against Rhizopus and A .flavus respectively which did not have potent activity even at a higher concentration of 200 μg/ mL. Compound 22, which has bulky bromo substitution at the para position of the phenyl rings attached to C-6 carbon of cyclohexenone moiety exerted good antifungal activity against all the tested fungal strains. All the naphthyl hydrazone derivatives 17-24 were more potent and exerted good antifungal activity when compared to the standard drug Fluconazole. Compounds with electron withdrawing fluoro, chloro, bromo or nitro substituent at the phenyl rings or compounds that having electron donating methyl or methoxy substitutents attached to C-6 of cyclohexenone moiety, exerted excellent activity against all the tested fungal strains.